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The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.
Article
The H+-translocating ATPase complex from the thermophilic bacterium PS3 (TF0-F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TF0). TF0 was obtained by treating TF0-F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TF0 contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TF0 vesicles). The binding of TF1 to TF0 present in vesicles restored energy-transducing activities, such as ATP-32Pi exchange, dicyclohexylcarbodiimide-sensitive ATPase, and ATP-dependent enhancement of 8-anilinonaphthalene-1-sulfonate fluorescence. Treatments such as protease digestion and chemical modification with acetic anhydride, succinic anhydride, or diazobenzenesulfonic acid destroyed the TF1-binding activity, which was caused by band 6 protein. Band 8 protein was a proteolipid that reacted specifically with dicylcohexyl-carbodiimide and seemed to play a central role in H+ conduction through the membrane.
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The polymorphic mitochondrial translation product var1 has been analyzed by one- and two-dimensional gel electrophoresis and by proteolytic cleavage of the radiolabeled product. Different apparent molecular weight forms of var1 ranging between 40,000 and 44,000 show “normal” migration behavior as a function of the acrylamide concentration on sodium dodecyl sulfate-polyacrylamide gels. Comparisons of peptide fragment patterns generated by digestion of different molecular weight forms of var1 with papain and a protease from Staphylococcus aureus V8 show considerable fragment homology; some partial fragments retain the molecular weight differences of the undigested product; others shown no homology and suggest the presence of unique cleavage sites. Analysis on a two-dimensional system consisting of electrophoresis in the first dimension on acid-urea gels and in the second dimension on sodium dodecyl sulfate-polyacrylamide gels, show that var1 behaves as a basic protein migrating between the cytoplasmic large subunit ribosomal proteins L2 and L3. Var1 is the only major labeled polypeptide species to be resolved in this two dimensional gel system when cells are labeled with 35SO42- in vivo in the presence of cycloheximide. By analyzing yeast strains containing different molecular weight forms of var1, we show that the protein is specifically associated with the 38 S mitochondrial ribosomal subunit. By Coomassie blue staining, it appears to be present in amounts roughly equivalent to the other proteins of the 38 S subunit and may be an integral ribosomal protein since it is not removed by a high salt wash. When cells are labeled in viva with 35SO42- in the absence of inhibitors, most, if not all, of the var1 is associated with the 38 S subunit. Between 20% and 80% of the var1 can be found in the postmitochondrial supernatant fraction. Marker enzyme distribution studies suggest that this “extra mitochondrial” var1 is released by mechanical damage of the mitochondria.
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Mutants of Neurospora crassa have been isolated that are highly resistant to inhibition by oligomycin, an inhibitor of mitochondrial ATPase activity. Dixon plots (Dixon, M., and Webb, E.C. (1964) Enzymes, 2nd Ed, pp. 328-330, Academic Press, New York) of oligomycin inhibition curves of the parent strain and the resistant mutants are linear, indicating that oligomycin interacts at a single site within the ATPase complex. The Ki values obtained from the mutants vary from 150 to 900 times greater than the Ki obtained for the parent strain. The parent strain and the oligomycin-resistant mutants are also inhibited by bathophenanthroline, a lipophilic chelating agent that inhibits F1 ATPase activity. Dixon plots of bathophenanthroline inhibition curves are also linear and Ki values obtained are all approximately equal. Crosses of the oligomycin-resistant mutants to the oligomycin-sensitive parent strain show a mendelian segregation of the resistance characteristic. These data show that mutations leading to oligomycin resistance in Neurospora are due to alterations in nuclear genes.
Article
A product of mitochondrial protein synthesis in rat liver mitochondria, characterized by a low molecular weight (Mr is less than 10000) and an unusually high hydrophobicity, has been identified as the dicyclohexylcarbodiimide-binding protein and as a peptide of the hydrophobic sector of the mitochondrial ATPase complex. The purified protein still possesses the ability of bind dicyclohexylcarbodiimide.
Article
A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.
Article
Fourteen stable lines of myeloma-spleen cell hybrids producing antibodies against the mitochondrial H+-ATPase have been isolated. One reacted with the α-subunit of the enzyme complex (Mr 56 000), nine with the β-subunit (Mr 54 000), and four with a 25 kDa subunit which has not been previously characterized. These antibodies are inhibitory or stimulatory or have no effect upon the enzyme activity. Two of the monoclonal anti-β-subunit antibodies were found to be particularly effective in immunoprecipitating intact H+-ATPase complex.
Article
Incubation of mitochondria from Neurospora crassa and Saccharomyces cerevisiae with the radioactive ATPase inhibitor [14C]dicyclohexylcarbodiimide results in the irreversible and rather specific labelling of a low-molecular-weight polypeptide. This dicyclohexylcarbodiimide-binding protein is identical with the smallest subunit (Mr8000) of the mitochondrial ATPase complex, and it occurs as oligomer, probably as hexamer, in the enzyme protein. The dicyclohexylcarbodiimide-binding protein is extracted from whole mitochondria with neutral chloroform/methanol both in the free and in the inhibitor-modified form. In Neurospora and yeast, this extraction is highly selective and the protein is obtained in homogeneous form when the mitochondria have been prewashed with certain organic solvents. The bound dicyclohexylcarbodiimide label is enriched in the purified protein up to 50-fold compared to whole mitochondria. Based on the amino acid analysis, the dicyclohexylcarbodiimide-binding protein from Neurospora and yeast consists of at least 81 and 76 residues, respectively. The content of hydrophobic residues is extremely high. Histidine and tryptophan are absent. The N-terminal amino acid is tyrosine in Neurospora and formylmethionine in yeast.
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A single gene nuclear yeast mutant was isolated whose mitochondrial F1-ATPase was resistant to the specific F1 inhibitor aurovertin. The mutant enzyme was not cross-resistant to other F1 inhibitors. The binding of aurovertin to F1 and to the two largest F1 subunits (alpha and beta) was measured by enhancement of aurovertin fluorescence. Aurovertin bound to wild type F1-ATPase and to its monomeric beta subunit with about the same binding constant. It failed to bind to wild type alpha subunit or to either F1 or F1 subunits from the mutant. The aurovertin-resistant mutant thus contains an altered nuclear gene which specifies the structure of the beta subunit of F1.
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The dicyclohexylcarbodiimide-sensitive ATPase from spinach chloroplast has been isolated. On sodium dodecyl sulfate gels, seven different polypeptides were seen. Five of these polypeptides coincided with the CF1 subunits, a 7,500-dalton peptide was identified as the proteolipid which interacts with [14C]dicyclohexylcarbodiimide, and there was a 15,500-dalton hydrophobic polypeptide with unknown function. In two-dimentional gels, two additional peptides were resolved, one 17,500 daltons (co-migrating in sodium dodecyl sulfate gels with subunit delta) and one 13,500 daltons (co-migrating with subunit epsilon). Reconstitution was obtained by freezing and thawing the complex with a crude mixture of phospholipids. After reconstitution the complex catalyzed 32P1-ATP exchange (rates of 200 to 400 nmoles x mg-1 x min-1) and ATP formation during acid-to-base transition. These reactions were inhibited by dicyclohexylcarbodiimide and uncouplers. Uncouplers at low concentrations stimulated and at high concentrations inhibited the Mg2+-ATPase activity. ATP hydrolysis and 32P1-ATP exchange were catalyzed by the complex in the presence of either Mg2+ or Mn2+ but not with Ca2+ or Co2+. ATP and GTP were substrates for the exchange reaction but not ADP or CTP.
Article
1. F1-ATPase has been extracted by the diphosphatidylglycerol procedure from mitochondrial ATPase complexes that differ in ATPase activity, cold stability, ATPase inhibitor and magnesium content. 2. The ATPase activity of the isolated enzymes was dependent upon the activity of the original particles. In this respect, F1-ATPase extracted from submitochondrial particles prepared in ammonia (pH 9.2) and filtered through Sephadex G-50 was comparable to the enzyme purified by conventional procedures (Horstman, L.L. and Racker, E. (1970) J. Biol. Chem. 245, 1336--1344), whereas F1-ATPase extracted from submitochondrial particles prepared in the presence of magnesium and ATP at neutral pH was similar to factor A (Andreoli, T.E., Lam, K.W. and Sanadi, D.R. (1965) J. Biol. Chem. 240, 2644--2653). 3. No systematic relationship has been found in these F1-ATPase preparations between their ATPase inhibitor content and ATPase activity. Rather, a relationship has been observed between this activity and the efficiency of the ATPase inhibitor-F1-ATPase association within the membrane. 4. It is concluded that the ATPase activity of isolated F1-ATPase reflects the properties of original ATPase complex provided a rapid and not denaturing procedure of isolation is employed.
Article
1. A cold-stable oligomycin-sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X-100 and purified by gel filtration. 2. The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000--6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme. 3. The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing. 4. The molecular weight as determined by gel filtration is about 480 000 +/- 30 000. S020,w is 1.45 +/- 0.1 S and the pI is 5.4. 5. The amino acid composition of the F0F1 complex is compared with the data obtained for the F1 moiety of the enzyme. 6. Quantitative data on the sensitivity to N,N'-dicyclohexyl-carbodiimide as well as kinetic parameters, regarding substrate specificity and dependence of ATPase activity on divalent cations, are reported.
Article
ATPase complex is isolated from mitochondria of Neurospora crassa by immunological techniques. The protein can be obtained rapidly and quantitatively in high purity by micro- or large-scale immunoprecipitation. Immunoprecipitation is applied to labeled and doubly labeled mitochondrial proteins to investigate the number and molecular weights of subunit polypeptides, the site of synthesis of subunit polypeptides, and the dicyclohexylcarbodiimide-binding protein. The ATPase complex obtained by large-scale immunoprecipitation is used as starting material for the isolation of hydrophobic polypeptides. The F1 moiety of the complex is solubilized by chloroform treatment of the mitochondria. The purified F1 protein is used to raise antibodies in rabbits. The whole ATPase complex is then precipitated by antiserum to F1 from mitochondria solubilized by Triton X-100. Mitochondria are isolated in the presence of the protease inhibitor phenylmethylsulfonyl fluoride. All operations are performed at room temperature.
Article
Selective toxicity is the basis of all successful therapeutic agents. The intrinsic similarity in the physiological, bioenergetic, and biochemical properties of energy transducing membranes of all living cells makes it particularly difficult to find agents acting upon membrane enzymes possessing any degree of selective toxicity. Few of the many inhibitors of membrane-bound magnesium-activated adenosine triphosphatases (Mg2+-ATPase) inhibitors described have ever been used therapeutically other than as antiseptics or for topical application. These compounds have proven to be invaluable tools in the study of membrane bioenergetics. Their use has led to an understanding of the function of Mg2+-ATPase of all living systems, particularly in the differentiation of the membrane-bound proton-translocating region of the enzyme from the site(s) of ATP hydrolysis and syntheses.
Article
Evidence is presented that a mitochondrial translation product (, 32 000) previously thought to be a subunit of the membrane sector of the yeast mitochondrial ATPase is a contaminant, consisting of subunit II of the cytochrome oxidase complex and cytochrome b apoprotein. Our data suggest that only two subunits (, 7600 and 20 000) of the mitochondrial ATPase are synthesized in the mitochondria.
Article
A water-soluble Mg2+-dependent ATPase (coupling factor F1) was isolated from the mitochondria of housefly thorax. It comprised about 14% of the proteins from a crude preparation. The F1 preparation was nearly homogeneous as assessed by gel electrophoresis, isoelectric focusing, and electron microscopy. It was composed of five subunits with the following apparent molecular weights: α, 68,000; β, 61,000; γ, 38,000; δ, 27,000; and ϵ, 17,500. The isoelectric pH (pI) of this protein was 7.3. F1 had a pH optimum of 8.2 and a temperature optimum between 37 and 45°C. The enzyme was fairly stable at 25°C. Nearly complete loss of activity was noticed at 0°C, while at 0 or 25°C, glycerol (20%) partially stabilized the enzyme activity against such inactivation. The Km value of the enzyme with respect to ATP was 0.4 mM. The activity was stimulated by low concentrations of 2,4-dinitrophenol. The enzyme was inhibited by azide, p-hydroxymercuribenzoate, and guanidine hydrochloride. Oligomycin and the pesticides pyrethrin, cyhexatin, and DDT have no effect on the enzyme activity. However, all of these chemicals inhibited intact Mg2+- ATPase. The results are discussed in the light of differential responses of soluble and intact ATPase to these pesticides.
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Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.
Article
In order to develop an eukaryotic vector with the Podospora plasmid, further characterization is required of the mitochondrial DNA into which this plasmid is integrated, a physical map (restriction sites) of the Podospora chondriome (size 95 kb) has been completed. As prerequisite for the establishment of a genetic (functional) map, 70% of the chondriome was cloned in E. coli vectors. Using mitochondrial genes from Saccharomyces cerevisiae, six structural genes were located on the Podospora chondriome by cross hybridization experiments. There is strong evidence that the plasmid is inserted into the cytochrome b gene. A comparison of the genetic map of the Podospora chondriome with those of Neurospora crassa and Aspergillus nidulans exhibits a rather good accordance with respect to the sequence of genes.
Chapter
The in vivo mechanism of assembly of the mitochondrion poses formidable conceptual and experimental challenges to the investigator. The morphological complexity of the organelle coupled with the fact that the synthesis of the constituent proteins and lipids occurs in two spatially separate compartments of the cell indicates a highly ordered process whose most general features remain unknown.
Chapter
Current interest in membrane biogenesis is focused on a series of questions that pertain to: the structure and regulation of genes for membrane proteins; the routes taken by membrane proteins and lipids from their sites of synthesis in the cell to their sites of insertion in the membrane; the coordination between the synthesis of membrane proteins and lipids; and the mechanisms by which proteins and lipids are inserted into the membrane bilayer so as to ensure its continued asymmetry. In considering the biogenesis of mitochondrial membranes, these questions must be extended to include, in addition, the respective roles played by mitochondrial and nuclear genomes in the assembly process. In this review we will first briefly summarize the coordination between nuclear and mitochondrial genomes in mitochondrial biogenesis and then discuss the pathways followed by nuclear and mitochondrial gene products into the genetic mosaic of the inner mitochondrial membrane.
Chapter
The energy transducing ATPase of mitochondria, chloroplasts, and bacteria is one of the most complex enzymes. It consists of at least ten different subunit polypeptides ranging in molecular weight from 60,000 to 8000 daltons. We have concentrated our efforts during the past few years on the smallest subunit of 8000 daltons, especially on its chemical characterization. This extremely hydrophobic polypeptide occurs in the complex as an oligomer, probably as a hexamer (Sebald et al., 1978) and is thus a major subunit, comprising about 10% of the total enzyme protein. Together with at least two further hydrophobic polypeptides it constitutes the membrane factor Fo (Sone et al., 1975; Sebald, 1977), which has been shown to exhibit the properties of a proton channel (Hinkle and Horstman, 1971; Okamoto et al., 1977).
Chapter
The transport of solutes into and out of the cytoplasm across the plasma and assorted endomembranes of fungal cells is essential for their survival. These fluxes predominantly occur by means of transport proteins and are required for such processes as the uptake of nutrients, maintenance of turgor, cell expansion, development, the compartmentation of potentially cytotoxic ions and signal transduction.
Chapter
The progress made in a certain scientific area does not proceed at a constant rate, but is subjected to large alterations. It appears to me that a remarkable step forward has been made in the field of mitochondrial biogenesis during the last two years, although this is not comparable to the period of “dramatic moves” which occurred in the early seventies. The aim of this chapter is not to give an extensive review on the numerous publications which have emerged recently, is not to include a volume of detailed facts, and is not to enter into the diversities found in the various organisms investigated. (This information is supplied thoroughly by the proceedings of recent symposia.) Rather, an attempt has been made to survey a selected number of experimental works of exemplary character and subject them to a critical appraisal.
Chapter
Mitochondria are organelles comprising a fundamental and characteristic feature of eukaryotic cytoplasm. The familiar basic structural theme of outer membrane enclosing a folded inner membrane is elaborated into an extensive variety of forms in different eukaryotic organisms and cell types. Inside living cells mitochondrial form is far from static, and harbored within these mobile, plastic, mitochondrial structures is an immense and varied repertoire of biochemical functions.
Article
When isolated cucumber (Cucumis sativus L.) mitochondria were treated with 14C-labelled dicyclohexylcarbodiimide (DCCD), a single polypeptide was predominantly labelled. This polypeptide was soluble in 1-butanol or chloroform: methanol (2: 1, v/v) and had an apparent molecular mass of approximately 7 kDa; it therefore had the characteristic properties of the DCCD-binding proteolipid subunit of the ATP synthase complexes of mitochondria, chloroplasts, and prokaryotes. When isolated cucumber mitochondria were allowed to synthesize protein in the presence of [35S]methionine and then extracted with 1-butanol or chloroform: methanol (2: l, v/v), a 35S-labelled proteolipid that migrated more rapidly on SDS-polyacrylamide gels than the pro-teolipid labelled by [14C]DCCD was solubilized. Treatment of mitochondria with unlabelled DCCD after they had been allowed to synthesize protein, specifically converted some of the [35S]methionine-labelled proteolipid to a form that comigrated with the [14C]DCCD-labelled proteolipid. We therefore conclude that a DCCD-binding proteolipid is synthesized by isolated cucumber mitochondria.
Article
We have determined the carbohydrate and lipid contents of vacuolar membranes fromNeurospora crassa, and have compared them to mitochondrial membranes, endoplasmic reticulum, and plasma membranes. These four membrane fractions were clearly distinct from each other in polypeptide composition, as judged by polyacrylamide gel electrophoresis. The vacuolar membranes proved unusual in two respects: the contained very high amounts of carbohydrate and were the only membranes with significant levels of phosphatidylserine. As in other eucaryotic cells, the mitochondrial membranes were unique in having high amounts of cardiolipin but virtually no sterol. Although the endoplasmic reticulum and plasma membranes were qualitatively similar to each other, the plasma membranes could be distinguished by a higher carbohydrate content, whereas the endoplasmic reticulum had a characteristically high ratio of phosphatidylcholine to phosphatidylethanolamine. The fatty acid compositions of all four membranes were similar, except that mitochondrial membranes contained about half as much saturated fatty acids as the other three fractions.
Article
Subunit 9 of mitochondriat ATPase (Su9) is synthesized in reticulocyte lysates programmed with Neurospora poly A-RNA, and in a Neurospora cell free system as a precursor with a higher apparent molecular weight than the mature protein (Mr 16,400 vs. 10,500). The RNA which directs the synthesis of Su9 precursor is associated with free polysomes. The precursor occurs as a high molecular weight aggregate in the postribosomal supernatant of reticulocyte lysates. Transfer in vitro of the precursor into isolated mitochondria is demon- strated. This process includes the correct proteolytic cleavage of the precursor to the mature form. After transfer, the protein acquires the following properties of the assembled subunit: it is resistant to added protease, it is soluble in chloroform/methanol, and it can be immunopre- cipitated with antibodies to FI-ATPase. The precursor to Su9 is also detected in intact cells after pulse labeling. Processing in vivo takes place posttranslationally. It is inhibited by the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). A hypothetical mechanism is discussed for the intracellular transfer of Su9. It entails synthesis on free polysomes, release of the precursor into the cytosol, recognition by a receptor on the mitochondrial surface, and transfer into the inner mitochondrial membrane, which is accom- panied by proteolytic cleavage and which depends on an electrical potential across the inner mitochondrial membrane.
Article
Earlier communications from this laboratory have shown that DDT inhibited oligomycin-sensitive Mg2+-ATPase (EC 3.6.1.3) but that its active component, F1, was not affected. In the present investigation evidence has been obtained to determine the nature of the requirements for DDT sensitivity. The results showed that DDT sensitivity was conferred to F1 from pig heart mitochondrial preparations when it was bound to F0 from the same preparation. The F1 from house fly (Musca domestica L) thorax was able to bind to F0 from pig heart. This combination showed similar sensitivity to that of the original F1-F0 combination from pig heart mitochondria. However, when F1 from pig heart mitochondria was incorporated into F0 depleted in oligomycin sensitivity-conferring protein (OSCP) from the same source, the resulting ATPase activity was insensitive to DDT. Addition of crude (50–200 μg) or purified (5–20 μg) OSCP in the above preparation restored DDT sensitivity. Presence of dioleyl or dipalmitoyl phosphatidyl choline or Triton X-100 in the reaction medium antagonized the DDT inhibitions. Depletion of phospholipids from submitochondrial membrane preparations (SMP) decreased ATPase activity. Addition of dioleyl or soybean phosphatidyl choline to this lipid-depleted preparation restored DDT sensitivity. Evidence presented suggests that DDT acted on F1 in association with one or more membrane components and that OSCP and phospholipid were essential for DDT sensitivity.
Article
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Abstract In the ten years since the proposal8 and evidence10 that translocation of secretory proteins across the endoplasmic reticular membrane depended upon an amino acid sequence “signal”, our understanding of the events which allow compartmentation of eukaryotic proteins into different organelles has progressed rapidly. During this time, segregation of bacterial proteins has also been found to share many similar features.2 While the proposal that all proteins destined to cross organellar15 membranes might utilize an identical mechanism for translocation has been disproved, the impetus provided by the “signal hypothesis” greatly accelerated investigation in this area. Present evidence indicates that intracellular translocation across membranes, i.e., compartmentation, can occur by four mechanisms 1. Removal of NH2-terminal signal (pre) peptide of 15 to 30 amino acids from secretory, some viral envelope, integral plasma membrane and lysosomal proteins occur when they cross the ER membrane during translation. Further co-translational and post-translational modifications including glycosylation, processing of oligosaccharide side chains, phosphorylation, further proteolytic processing, acetylation, amidation and fatty acylation also play a role in determining the ultimate localization of these newly synthesized proteins 2. Alternatively, for ovalbumin and some ER integral membrane proteins, compartmentation occurs during translation but without the removal of any peptide 3. For most chloroplast and mitochondrial proteins, translocation from the cytoplasm into the organelle occurs after translation is completed and involves the removal of an NH2-terminal peptide. In contrast to secretory proteins, however, this transit peptide is larger and probably hydrophilic 4. Finally, a few proteins destined for either the ER membrane, mitochondria, or peroxisomes are incorporated post-translationally but without the removal of any peptide While compartmentation has been shown to occur by these mechanisms, the component parts of the translocation apparatus are not well characterized. One of the major directions of current investigations is to separate, purify and characterize these components and, in most cases, proteases. In vitro reconstitution of the translocation event is essential for elucidating the mechanism of this reaction. A second approach, which has already provided useful information, is to construct genes with modifications in the coding regions of translocated proteins and to insert such genes into the bacterial genome. An alternative method is to introduce amino acid analogs into the proteins and assess alterations in translocation. In conjunction with these studies, it is clear that further structural analysis of larger precursors, particularly of mitochondrial protein subunits, will be essential. When the above information is available, questions concerning the unique structural features of proteins which direct them to a specific organelle, and the components of the translocation apparatus which are common among different organelles can then be answered. Finally, the importance of compartmentation as a regulatory event in modifying localization of active proteins can then be addressed.
Article
Die wichtigste Energiequelle für alle in lebenden Organismen vorkommenden endergonischen Prozesse ist die Energie der Phosphorsäureanhydrid-Bindung der Nucleosidtriphosphate, speziell des Adenosintriphosphats (ATP). In aeroben Organismen, zum Beispiel Säugetieren, wird mehr als 90% des ATP in einem oxidative Phosphorylierung genannten Vorgang gebildet. Wie bei der Muskelkontraktion und der Nervenerregung bedient sich die Natur auch bei der oxidativen Phosphorylierung vektorieller Prozesse, die an einer Membran ablaufen, welche verschiedene Räume voneinander abgrenzt. Der vorliegende Übersichtsartikel befaßt sich mit der Funktion einer Reihe von wasserunlöslichen Membranproteinen und Enzymen, die vektoriell Elektronen sowie Protonen und andere Ionen transportieren und letztendlich zur Bildung von ATP führen. Die Maschinerie, welche die Substratoxidationsenergie in chemische Energie in Form der Phosphorsäureanhydrid-Bindung von ATP umwandelt, arbeitet mit sehr hohem Wirkungsgrad. In diesem Beitrag werden Struktur und Funktion des Systems der oxidativen Phosphorylierung in Mitochondrien behandelt. Das Gesamtsystem besteht aus der Elektronentransportkette, der ATP-Synthetase, der Adeninnucleotid-Translokase und dem Phosphat-Transportsystem. Die Elektronentransportkette kann in vier Multiprotein-Komplexe - an drei davon findet eine Energieumwandlung statt - und in die Elektronenüberträger Ubichinon und Cytochrom c unterteilt werden. Die bei der Substratoxidation freigesetzte Energie wird in chemische Energie in Form von ATP umgewandelt, wobei ein elektrochemischer Protonengradient als Zwischenform auftritt. Die energetischen Aspekte dieser Prozesse lassen sich mit der linearen irreversiblen Thermodynamik behandeln. Die letzten Jahre haben große Fortschritte bei der strukturellen Charakterisierung der beteiligten Proteine gebracht. Die Funktion der genannten Systeme ist teilweise auf molekularer Ebene erforscht; dies gilt insbesondere für den Protonen- und Adeninnucleotid-Transport sowie die ATP-Bildung.
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The energy transformation systems of Halobacterium halobium, Vibrio succinogenes, chloroplasts and mitochondria are described on the basis of the chemiosmotic theory. Nature seems to utilize three quite different mechanisms for electrogenic proton transport. Structure and function of the ATP-synthetase complexes, however, seem to be quite similar in the various energy-conserving systems.
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The ATP synthetase of Escherichia coli K12 was purified by a simple procedure. The dicyclohexylcarbodiimide-sensitive ATPase activity was enriched 21-fold. The ATP synthetase preparation contained the eight polypeptides (alpha, beta, gamma, a,delta, b,espilon, c) of the enzyme and a residual contamination (4% of the total protein) as shown by dodecylsulfate/polyacrylamide electrophoresis. The polypeptide c was specifically labelled with [14C]dicyclohexylcarbodiimide. Energy-transducing activities were reconstituted from soybean phospholipids and the purified enzyme. The proteoliposomes exhibited a significantly higher ATP-32Pi exchange activity and a higher proton-translocating activity as compared to the untreated membranes.
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The products of mitochondrial protein synthesis in yeast have been studied by analytical gel electrophoresis. Over 60% of the total counts incorporated into mitochondria when cytoplasmic protein synthesis is blocked are associated with five proteins which are separated on acrylamide gels in the presence of sodium dodecyl sulfate. The mitochondrial products have been found to be extracted from the membrane with acidic chloroform-methanol. Under neutral conditions chloroform-methanol removes predominantly a protein which has been estimated to have a molecular weight of 7,800. This protein has been found to be present in the membrane in a polymeric form with an apparent molecular weight of 45,000. Conversion of the polymer to the low molecular weight form is achieved by treatment of mitochondrial membranes with chloroform-methanol or by depolymerization in sodium dodecyl sulfate at alkaline pH. The low molecular weight protein appears to be the major product of mitochondrial protein synthesis. Its possible role in mitochondrial biogenesis is discussed.
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Earlier studies from this laboratory have shown that cytochrome c oxidase from bakers' yeast consists of seven polypeptides. The three largest ones (molecular weights 42,000, 34,500, and 23,000) are synthesized on mitochondrial ribosomes, whereas the four smallest ones (molecular weights 14,000, 12,500, 12,500, and 9,500) are synthesized on cytoplasmic ribosomes. In order to study the assembly of the functional oligomeric enzyme, four cytochrome c oxidase-less mutants of the Saccharomyces cerevisiae strain D273-10B were analyzed for residual cytochrome c oxidase polypeptides. Three were nuclear mutants specifically deficient in cytochrome c oxidase; the fourth mutant was an extrachromosomally inherited petite strain lacking not only cytochrome c oxidase but several other mitochondrial components as well. Cytochrome c oxidase polypeptides in the respiration-deficient mitochondria were identified by immunodiffusion and by electrophoretic analysis of radioactively labeled immunoprecipitates. Each of the three nuclear mutants lacked at least one cytochrome c oxidase component which, in the wild type, is synthesized on mitochondrial ribosomes. Mutant pet 494 was devoid of the 23,000-dalton polypeptide, whereas mutants pet E11 and pet 1030 were almost completely deficient in all three large cytochrome oxidase polypeptides. All nuclear mutants possessed near normal amounts of the cytoplasmically synthesized cytochrome c oxidase components. The extrachromosomal petite mutant lacked not only the three mitochondrially synthesized polypeptides, but also the 9,500-dalton polypeptide which is a product of cytoplasmic protein synthesis. The remaining cytochrome oxidase subunits were only loosely bound to the mitochondrial membrane, differing in this respect from the corresponding subunits in wild type mitochondria. These results permit the following conclusions. 1. The polypeptide of molecular weight 23,000 is apparently an essential component of cytochrome c oxidase, since its absence in mutant pet 494 is paralleled by a specific loss of the functional enzyme. 2. The mitochondrially synthesized cytochrome c oxidase subunits are necessary for the tight binding of the cytoplasmically synthesized cytochrome c oxidase subunits to the mitochondrial inner membrane. 3. The synthesis (or intregration) of mitochondrially synthesized cytochrome c oxidase subunits can be prevented by nuclear mutations. Conversely, extrachromosomal mutations can impair or completely prevent the integration of cytoplasmically synthesized cytochrome c oxidase subunits. These experiments also showed that, under our conditions, the three large subunits of cytochrome c oxidase account for 20 to 25% of the protein synthesized by mitochondria.
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Eighteen respiration-deficient yeast mutants were isolated from wild type cells after mutagenesis with ethylmethane sulfonate. Segregation analysis indicated that each mutant phenotype resulted from the mutation of a single nuclear gene. All mutants except one efficiently retained functional mitochondrial DNA as measured by ability to complement mitochondrial DNA-less tester strains. When the mutants were crossed pairwise and the resulting zygotes checked for functional respiration, the 18 strains could be classified into seven complementation groups. Three of these groups were characterized by a specific loss of cytochrome aa3. Another group included pleiotropic mutants which lacked cytochrome aa3, b, and c1, but not oligomycin-sensitive mitochondrial ATPase. Still another group was represented by a pleiotropic mutant that was not only deficient in cytochromes aa3, b, and c1 but in mitochondrial ATPase as well. The two remaining complementation groups included mutants that were completely deficient in cytochrome aa3 and partially deficient in other mitochondrial constituents. All of the mutants still exhibited mitochondrial protein synthesis. However, when the proteins synthesized by the mutant mitochondria in vivo were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, they usually lacked several species made by wild type mitochondria. These results show that nuclear mutations can affect either the synthesis of mitochondrial translation products or their integration into the mitochondrial inner membrane. Since mitochondrial protein synthesis can still be detected in single gene mutants with multiple mitochondrial deficiencies, we suggest that some nuclear genes may code for mitochondrial “organizer” proteins that control the correct assembly of the mitochondrial inner membrane.
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The rutamycin-sensitive ATPase complex of yeast mitocondria consists of the ATPase, F1, of an easily extractable protein (OSCP) which is concerned with the binding of F1 to the membrane, and of another membrane factor which contains at least four distinct subunit proteins. When glucose-repressed yeast are incubated in a low glucose medium containing cycloheximide and radioactive leucine, label is incorporated into a fraction which forms a precipitable complex with antiserum to the rutamycin-sensitive ATPase. Analysis of the antibody precipitate by gel electrophoresis has revealed that at least four distinct proteins are labeled. The labeled products comigrate with known subunits of the rutamycin-sensitive ATPase and comprise those protein components of the ATPase which are most firmly associated with the membrane. These results indicate that with the exception of F1 and OSCP, which are synthesized in the cytoplasm, the remaining subunits of the ATPase are made by the mitochondrion.
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The rutamycin-sensitive adenosine triphosphatase (ATPase) complex has been solubilized from yeast submitochondrial particles with Triton X-100 and further purified by centrifugation on a glycerol gradient. The purified enzyme is dispersed and by electron microscopic criteria appears to be an oval-shaped globular particle 100 by 150 A. Both the cold-labile ATPase, coupling factor 1 (F1) and the rutamycin-sensitive complex have been analyzed for their subunit protein composition and their molecular weights by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of these analyses indicate that F1 contains five different subunit proteins. The two major subunits of F1 have molecular weights of 58,500 and 54,000. The rutamycin-sensitive complex contains all of the components of F1 and four additional proteins. The composite molecular weight of the proteins specific to the complex is approximately 100,000. Two lines of evidence indicate that the subunit proteins seen on polyacrylamide gels are intrinsic components of the complex. The ATPase complex reconstituted from F1, oligomycin sensitivity-conferring protein, and depleted membranes has been shown to contain all of the subunits seen in the native enzyme. Secondly, antibody prepared against F1 cause the nine subunits of the complex to precipitate.
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The rutamycin-insensitive ATPase (F1) of yeast mitochondria was found in previous studies to be synthesized on the cytoplasmic ribosomes and to accumulate in the postribosomal supernatant (Tzagoloff, A. (1969) J. Biol. Chem. 244, 5027). The enzyme has been purified from the postribosomal fraction of yeast grown in the presence of chloramphenicol and compared with F1 isolated from normal yeast mitochondria. The subunit protein compositions of the two preparations are identical, indicating that all of the component proteins of F1 are products of the cytoribosomal system of protein synthesis. Following removal of chloramphenicol and further incubation of the cells in growth medium (derepression), an increase of the ATPase activity of mitochondria and a loss of F1 activity in the postribosomal supernatant are observed. Evidence is presented indicating that during derepression the postribosomal F1 is incorporated into the larger rutamycin-sensitive ATPase complex of mitochondria.
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The isolated subunits of beef heart mitochondrial ATPase were characterized with regard to physical and chemical properties. Three subunits, molecular weight 54,000, 50,000, and 33,000, respectively, constituted 93% of the protein of the oligomer. The remainder of the molecule consisted of two subunits of molecular weight 17,300 and 5,700. The latter peptide (Subunit 5) was identified by physical criteria with the ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. (1963) J. Biol. Chem. 238, 3762–3769). Subunit 5 was a basic protein (pI = 10.4) which was removed from the ATPase in the form of a mixture of monomer (5,700) and dimer (11,350). Conversion of the dimer to the monomer was dependent on the presence of agents capable of reducing disulfide bonds.
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Purified cytochrome c oxidase from bakers' yeast can be resolved into six polypeptide bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weights of these components are I, 42,000; II, 34,500; III, 23,000; IV, 14,000; V, 12,500; and VI, 9,500. (Although Component V actually consists of two distinct polypeptide species, it will be regarded as homogeneous in this study.) In order to study the biosynthesis of these components, yeast cells were labeled with [³H]leucine in the presence of specific inhibitors of mitochondrial and cytoplasmic protein synthesis. Labeled cytochrome c oxidase components were then isolated from crude mitochondrial extracts by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of the three large Components I, II, and III was insensitive to cycloheximide and sensitive to erythromycin. Labeling of the two largest Components I and II was dependent on the presence of oxygen. The three small Components IV, V, and VI were not labeled in the presence of cycloheximide but became labeled in the presence of erythromycin. These results show that our cytochrome c oxidase preparation contains three polypeptides which are translated on mitochondrial ribosomes and three polypeptides which are translated on cytoplasmic ribosomes. Two of the mitochondrially synthesized polypeptides are only made in the presence of oxygen.
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A protein with the same properties as the oligomycin sensitivity-conferring protein (OSCP) of bovine heart mitochondria has been purified from yeast mitochondria. This protein stimulates the binding of F1 to extracted membranes by as much as 10-fold. The reconstituted particulate ATPase is inhibited by rutamycin. The yeast OSCP by itself does not form a complex with F1 nor does it confer rutamycin sensitivity on F1 in the absence of the membrane. The function of OSCP appears to be closely related to the binding of F1 to another component of the ATPase which is present in the membrane. The postribosomal supernatant of yeast cells derepressed on low concentrations of glucose in the presence of chloramphenicol has been shown to contain soluble OSCP. The amount of OSCP present in the cytoplasmic fraction is sufficient to bind all of the soluble F1 also present in this fraction. It is concluded that both OSCP and F1 are synthesized by the cytoplasmic-ribosomal system of the cell.
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Yeast cytochrome oxidase has been highly purified by a procedure involving solubilization in deoxycholic acid, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Standard preparations of beef cytochrome oxidase have been further purified by glycerol gradient centrifugation. The heme contents of the yeast and beef enzymes, respectively, were 15 and 12 nmoles of heme a per mg of protein. Yeast oxidase is composed of at least seven polypeptides when examined on polyacrylamide gels in the presence of sodium dodecyl sulfate. The molecular weights of the component polypeptides on 7.5% acrylamide gels range from 35,000 to 8,600. The beef enzyme has six subunits of molecular weights 34,500 to 7,300. The molecular weight of yeast cytochrome oxidase, determined by a combination of sucrose gradient centrifugation and gel filtration, was 226,000 after correction for bound detergent.
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A group of oligomycin-resistant mutants of Saccharomyces cerevisiae have been selected. Most are cytoplasmically inherited and have altered ATPase sensitivity in isolated mitochondria. Two mutants, resistant to growth on oligomycin, are not resistant when ATPase is tested in isolated mitochondria. One of these mutants shows normal Mendelian inheritance while the other is cytoplasmic. Studies on the isolated soluble ATPase enzyme from wild type cells show that the solubilized enzyme is not inhibited by oligomycin but the reconstituted enzyme complex is sensitive. ATPases from oligomycin-resistant mutants can also be separated into their component parts and subsequently reconstituted. The reconstituted enzyme complex resembles the mitochondrial-bound enzyme in its resistance to oligomycin inhibition. Preparation of hybrid reconstituted ATPase complexes from mixed components of the mutant and wild type enzymes shows that the resistance is contributed by the membrane fraction of the mutant ATPase. Thus, the mitochondrial DNA has been shown to direct the synthesis of a membrane component influencing the activity of the mitochondrial ATPase.
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A respiration-deficient nuclear mutant of Saccharomyces cerevisiae was isolated that lacked mitochondrial adenosine triphosphatase (F1). The absence of this enzyme was established by adenosine triphosphatase assays, by antibody-binding studies, and by radioimmunochemical tests for F1 subunits. The mutant also lacked mitochondrial ³²Pi-ATP exchange activity and exhibited greatly reduced amounts of cytochromes aa3, b, and c1. It possessed a mitochondrial protein-synthesizing system, but several products of this system were either abnormal or missing. Thus, the level of the three mitochondrially synthesized subunits of cytochrome c oxidase was at least 10 times lower than in the wild type. The mutant was unable to grow anaerobically even if the growth medium contained a fermentable substrate and unsaturated lipids. This property has not yet been reported for any S. cerevisiae strain. The mutant had a great tendency to lose its mitochondrial genome. The conversion to the double mutant state decreased the growth rate by a factor of 20 to 30. These results suggest that mitochondrial adenosine triphosphatase has an essential function even in nonrespiring cells. The mutant described here offers new possibilities for studying the assembly of F1 from its subunits and the role of F1 in mitochondrial biogenesis.
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Yeast cells grown under conditions of glucose repression (5.4% glucose) exhibit a lower ATPase activity than cells grown on 0.8% glucose. The ATPase activity of the mitochondria increases during derepression, and this increase can be shown to be accompanied by an increase in the F1 (ATPase) content of the mitochondrial membranes. The increase of ATPase in the mitochondrial fraction during derepression is prevented by chloramphenicol. Under these conditions, however, there is an accumulation of soluble ATPase in the postribosomal supernatant. The soluble ATPase has been partially purified, and its properties indicate it to be identical to F1. Cycloheximide also prevents the increase of ATPase activity in the mitochondrial fraction during derepression. There is no accumulation of ATPase in the postribosomal fraction of cells incubated in a derepression medium containing cycloheximide. These results are interpreted to indicate that F1 is synthesized by the cytoplasmic-ribosomal protein-synthesizing system of the yeast cell.
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1. The properties of a mitochondrial fraction (F0) which confers oligomycin sensitivity on soluble ATPase were found to resemble in several aspects the properties of particles that catalyze oxidative phosphorylation. Susceptibility to oligomycin, Dio-9, tri-n-butyltin chloride, detergents, and phospholipase A are shared by both systems. 2. Treatment of F0 with phospholipase A resulted in complete loss of activity. About half of the original activity was restored by treatment of the preparation with serum albumin and phospholipids. 3. Brief exposure of particles containing F0 activity to trypsin or to sonic oscillation either at pH 10.0 or in the presence of phospholipid resulted in partial loss of F0 activity. Full restoration of activity was achieved by addition of coupling factor 4.
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1. A Mg⁺⁺-dependent adenosine triphosphatase was solubilized and purified from bakers' yeast mitochondria. The enzyme resembled mitochondrial ATPase from beef heart with respect to substrate specificity, cold lability, and other physical properties. 2. An antiserum against the purified yeast enzyme inhibited the ATPase activity of the soluble enzyme as well as ATPase and oxidative phosphorylation in submitochondrial yeast particles. Mitochondrial ATPase from beef heart or from Neurospora crassa was not inhibited by the antiserum. 3. Submitochondrial beef heart particles devoid of endogenous ATPase could bind the purified yeast enzyme without changing its immunological specificity. The ATPase activity of the resultant “hybrid” particles, like that of beef heart particles, was strongly inhibited by low levels of rutamycin. In contrast, submitochondrial particles from yeast were much less sensitive to this inhibitor. 4. The yeast enzyme stimulated oxidative phosphorylation in beef heart particles which were deficient in, but not devoid of, endogenous ATPase. The stimulation was dependent on the presence of beef heart coupling factor 1 (F1) in these particles and was unaffected by the antiserum against the yeast enzyme. Antiserum against beef heart F1 strongly inhibited phosphorylation. These results suggest that yeast F1, in contrast to beef heart F1, does not significantly participate in phosphate transfer reactions when it functions as a coupling factor in beef heart particles. Rather, it is proposed that the stimulation by yeast F1 is due to an effect on the membrane structure.
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1. A factor conferring oligomycin sensitivity on mitochondrial ATPase was obtained from submitochondrial particles by sequential treatment with trypsin and urea, and was purified by fractionation with ammonium sulfate in the presence of 2% cholate. The preparation had a very low content of phospholipids and respiratory enzymes. 2. By omitting treatment with urea, an insoluble, oligomycin-sensitive ATPase was isolated which was also low in respiratory enzymes and phospholipids. 3. Addition of soluble mitochondrial ATPase to the factor conferring oligomycin sensitivity resulted in a marked inhibition of ATPase activity, which was fully restored by addition of phospholipids. Similarly, the isolated oligomycin-sensitive ATPase required phospholipids for ATPase activity. 4. The interaction of these preparations with tritium-labeled ATPase, ¹⁴C-phospholipids and ¹⁴C-rutamycin was analyzed quantitatively. The binding capacity of the factor for ³H-labeled ATPase was much larger than that of various particles containing phospholipids. Addition of phospholipids interfered with the binding of ³H-labeled ATPase. Inhibition by ¹⁴C-rutamycin was reversed by repeated washing of particles with phospholipids.
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1. A mitochondrial fraction from beef heart (TU-particles) was shown to confer rutamycin sensitivity to added mitochondrial ATPase (F1). After sonic oscillation at an alkaline pH, the resulting particles (TUA-particles) no longer had this activity. A rutamycin sensitivity factor (Fc), purified from crude preparations of coupling factor 4, restored the capacity of the particles to confer rutamycin sensitivity to F1. Highly purified preparations of coupling factor 5 contained Fc, and the relationship between Fc and F5 is discussed. 2. The binding of F1 to TUA-particles was shown to take place with Mg⁺⁺ (0.8 mM) alone, but the bound ATPase was insensitive to rutamycin. Other divalent cations were less effective at these low concentrations; higher concentrations of monovalent cations (20 to 50 mM) substituted for Mg⁺⁺. The particulate ATPase activity was rendered rutamycin-sensitive by addition of Fc. 3. Exposure of either TUA-particles or Fc to heat or to trypsin resulted in complete loss of rutamycin sensitivity of the reconstituted system. This finding indicates that two labile factors contribute to the phenomenon. In contrast to the lability of the individual components, the Fc-TUA-F1 particle complex was resistant to heat and trypsin treatment under the same conditions. This observation represents another example of allotopy. 4. Dicyclohexylcarbodiimide was shown to inhibit particle-bound ATPase in a manner similar to rutamycin, except that no reversibility could be demonstrated. The site of action of dicyclocarbodiimide was shown to be associated with the particles and not with Fc.
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Mitochondria of the respiration-deficient cytoplasmic “petite” mutant of Saccharomyces cerevisiae contain adenosine triphosphatase. The enzyme associated with the mutant mitochondria is cold-labile and insensitive to oligomycin. In contrast, ATPase bound to mitochondria of the wild type strain is cold-stable and strongly inhibited by oligomycin. The mitochondrial ATPase of the “petite” mutant was purified and compared with the corresponding enzyme isolated from wild type cells. The two enzyme preparations were found to be indistinguishable with respect to enzymic properties, sedimentation coefficient, and immunological specificity. The purified mitochondrial ATPase from the “petite” mutant was bound to ATPase-deficient submitochondrial particles prepared from bovine heart mitochondria. The yeast enzyme was thereby rendered cold-stable and sensitive to oligomycin. It is concluded that the mitochondrial ATPase itself is not altered in the “petite” mutant. The unusual properties of the enzyme associated with the mutant mitochondria thus reflect an impairment of its linkage to the inner mitochondrial membrane.
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Mitochondrial particles prepared from glucose-repressed yeast bind only nominal amounts of the soluble ATPase components, rutamycin-insensitive ATPase and oligomycin sensitivity-conferring protein, when tested in an in vitro reconstitution assay. The capacity of the membranes to bind these components and to reconstitute rutamycin-sensitive ATPase increases when the cells are incubated in a derepression medium, suggesting that a membrane factor essential for the reconstitution is synthesized during the derepression. The effect of inhibitors of protein synthesis on the increase of the membrane factor during derepression has been studied. The membrane factor does not increase when either chloramphenicol or cycloheximide is added to the derepression medium. Increase of membrane factor, however, is found when yeast are incubated sequentially in derepression media containing first chloramphenicol and then cycloheximide. These results have been interpreted to indicate that the membrane factor is made by the mitochondrial protein-synthesizing system and that its synthesis is stimulated by products of the cytoplasmic ribosomal protein-synthesizing system. A positive control of mitochondrial protein synthesis by products of cytoplasmic ribosomal system is also supported by studies on the in vivo incorporation of amino acids into mitochondrial membrane proteins.
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Homogeneous mitochondrial ATPase from rat liver binds ADP in a rapidly reversible manner. The enzyme-bound ADP can be recovered quantitatively as ADP. In the absence of Mg2+, the enzyme exhibits 0.88 binding sites for ADP per enzyme molecule with an intrinsic dissociation constant of 0.94 µm. In the presence of Mg2+, the enzyme loses 90% of its ATPase activity but does not lose the ability to bind ADP. Short term binding experiments detect 0.65 binding sites per enzyme molecule with an intrinsic dissociation constant of 2.1 µm. The Km (ADP) for oxidative phosphorylation catalyzed by purified inner membrane vesicles, in which mitochondrial ATPase is located on the outer surface of the membrane directly available to added ADP, was found to be 3.8 µm. Aurovertin, a potent inhibitor of oxidative phosphorylation, inhibits binding of ADP by mitochondrial ATPase in the absence of Mg2+ in a manner similar to its inhibition of oxidative phosphorylation. In the presence of Mg2+, however, binding is enhanced by aurovertin. Taken together, the results show that the binding of ADP by soluble mitochondrial ATPase has important properties in common with the interaction of ADP with the functional oxidative phosphorylation system.
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1. A protein which inhibits mitochondrial ATPase has been isolated from bovine heart mitochondria in pure form and its properties have been characterized. 2. To obtain inhibition it was necessary to incubate the protein with ATPase in the presence of Mg++ and ATP. The specificity of this requirement for cation and nucleotide was similar to that reported previously for ATPase activity. About 4 times as much inhibitor was required to inhibit soluble ATPase as compared to particle-bound ATPase. 3. Soluble ATPase purified from a mitochondrial extract prepared with a Nossal shaker as described previously contained considerable amounts of ATPase inhibitor. A new simple procedure for the purification of ATPase was developed starting with an extract obtained from mitochondria by sonic oscillation. A second, more complex procedure was developed starting with an extract made from submitochondrial particles that had been depleted of inhibitor. The first preparation still contained inhibitor, although less than previously described preparations. The second preparation was virtually free of inhibitor. 4. Exposure of soluble ATPase to trypsin or chymotrypsin resulted in the removal of residual inhibitor and in a slight increase in ATPase activity. This treatment abolished coupling activity although the ATPase would still bind to the particles and its activity was partially sensitive to rutamycin.
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1. Amorphous membrane fragments depleted in P-lipids and cytochrome oxidase were isolated from bovine heart mitochondria and were reconstituted with P-lipids and coupling factors to yield vesicular structures. These vesicles catalyzed a 32Pi—ATP exchange and showed an induced enhancement of anilinonaphthalene sulfonate fluorescence on addition of ATP 2. 32Pi—ATP exchange and fluorescence enhancement were abolished by uncouplers of oxidative phosphorylation and by energy transfer inhibitors. The ATPase activity was inhibited by energy transfer inhibitors, but stimulated by uncouplers or by the combined action of nigericin and valinomycin in the presence of K+. Both ATPase activity and 32Pi—ATP exchange were inhibited by a specific antibody against coupling factor 1. 3. It was shown that the reconstitution of vesicular structures with functional activity required several hours. Rapid reconstitution resulted in inactive vesicles. Evidence for the formation of new vesicles from solubilized P-lipids was obtained by demonstrating inclusion of macromolecules such as 14C-labeled inulin or ferritin which could not be removed by washing.
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Cytochrome aa3 was purified 35- to 40-fold from submitochondrial particles of commercial bakers' yeast. The purification procedure involved solubilization of the enzyme with cholate, fractionation with ammonium sulfate, and chromatography on DEAE-cellulose in the presence of Triton X-100. The purified, active enzyme contained approximately 10 nmoles of heme a per mg of protein and was free of other hemoproteins. Upon sucrose gradient centrifugation in the presence of Triton X-100, it sedimented as a single peak with an apparent molecular weight of 190,000 to 225,000. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six major polypeptide bands of apparent molecular weight 42,000, 34,500, 23,000, 14,000, 12,500, and 9,500. The six components could be specifically precipitated from a crude mitochondrial extract with rabbit antiserum against the holoenzyme. The six components behaved as a single species during DEAE-cellulose chromatography and sucrose gradient centrifugation, and all could be precipitated with an antiserum against the three small components only. These and other observations indicate that all six components are physically associated with cytochrome c oxidase, even after solubilization of the mitochondrial inner membrane.
Chapter
A crucial development in the study of mitochondrial adenosine triphosphatase (ATPase) and its relationship to oxidative phosphorylation was the isolation by Pullman of a soluble ATPase (F1) that increased the efficiency of phosphorylation in certain types of submitochondrial particles. These studies provided the first direct evidence for the participation of the ATPase in oxidative phosphorylation and pointed out the usefulness of the tactic of resolution and reconstitution as an experimental approach to the study of the coupling mechanism. Although the soluble ATPase had the hallmarks of being the same enzyme that functions in uncoupled mitochondria, it differed from the latter in several significant respects. In contrast to the membrane-bound ATPase, which was inhibited by oligomycin and was stable at low temperatures, purified F1 was completely insensitive to oligomycin and was rapidly inactivated in the cold. More recently, the availability of the purified preparations of the oligomycin-sensitive ATPase complex has allowed characterization of its subunit proteins and in some instances, a definition of their function. Studies on the mitochondrial ATPase complex have helped to clarify some aspects of the coupling factors of oxidative phosphorylation, particularly their relationship to the subunit components of the complex and their function in energy transduction. Apart from its specific role in the terminal reactions of oxidative phosphorylation, the ATPase system has been of interest from the more general standpoint of the biochemistry of membrane enzymes.
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A number of biologically active glutarimide derivatives have been isolated from various streptomycetes. The derivatives for which chemical structures have been determined include cycloheximide, naramycin B, isocycloheximide, streptimidone, acetoxycycloheximide, inactone and the streptovitacins. All have in common the β (2-hydroxyethyl) glutarimide moiety attached to a cyclic or acyclic ketone. Although the antibiotics are noted particularly for their antifungal properties, they are toxic to a broad spectrum of organisms. Yet, a highly interesting characteristic of these antibiotics is the marked difference in their activity toward closely related organisms.
Article
The specific mitochondrial ATPase inhibitor protein has been examined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and the molecular weight was determined to be 10,500. This protein was present in some preparations of purified mitochondrial ATPase, but was completely absent from others. This difference was related to the technique of purification. The data suggest, therefore, that this protein is an easily dissociated subunit of mitochondrial ATPase. No difference was noted, however, in coupling efficiency between inhibitor-free and inhibitor-containing ATPase preparations in reconstitution experiments and the function of the inhibitor remains unclear.
Article
Two selective procedures are compared in an effort to isolate variants of mouse L cells containing structural gene mutations. Among the resulting variant cloned cell lines two types of alteration are found in the enzyme hypoxanthine phosphoribosyl transferase (EC 2.4.2.8.): enzyme with altered kinetic constants causing in vivo and in vitro resistance to 8 azaguanine: and enzyme with altered heat sensitivity in vitro. These results support the view that tissue culture cell variants can arise from structural gene mutations.
Article
1Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit−mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit− mutants with some but not other isonuclear Q− mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit− mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit−× mit+ crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit−, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PARI loci were used for this purpose.2The three methods when applied to the study of 48 mit− mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied.3The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drugresistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit− mutants and a large number of Q− clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers.4All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.
Article
1.1. F1 and OSCP, soluble coupling factors isolated from beef-heart mitochondria, form a complex.2.2. The binding between F1 and OSCP is weakened by (NH4)2SO4, but not enough to allow complete separation of F1 and OSCP in a linear sucrose gradient. Complete separation is achieved when both (NH4)2SO4 and deoxycholate are present.3.3. F1·X, a coupling factor also isolated from beef-heart mitochondria, can be separated in F1 and OSCP activities in the presence of (NH4)2SO4 and deoxycholate by centrifugation in a linear sucrose gradient or by gel filtration on a Sephadex G-100 column.4.4. F1·X preparations of low specific activity contain a contaminating protein. This protein can be dissociated from the complex by (NH4)2SO4. F1·X preparations with a high specific activity are virtually free of this contaminating protein.5.5. F1·X preparations contain approximately 30% free F1.6.6. Analytical gel electrophoresis of F1·X, F1 and OSCP in the presence of sodium dodecyl sulphate indicates that apart from F1 and OSCP no other components are present in pure F1·X.
Article
1. Mitochondria were isolated from a cytoplasmic respiratory-deficient variant of yeast Saccharomyces carlsbergensis NCYC 74.2. Mg2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of the mitochondrial fraction displayed two pH optima, at pH 6.2 and 9.5. The activity at pH 6.2 corresponded closely to that found in non-mutant yeast. The specific activity of the reaction at pH 9.5 was lower than in wild-yeast mitochondria and, unlike wild-type yeast ATPase, was resistant to oligomycin inhibition.3. The possibility is discussed that the ATPase activity of the mutant mitochondria may represent a preserved part of the oxidative phosphorylation system either structurally modified or lacking a component which would make it oligomycin sensitive.
Article
Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETRin vivo. VENR OLYR mutants show a high degree of resistance to venturicidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex.
Article
1. Two loci O Iand O IIlocalized on the mitochondrial DNA and conferring oligomycin resistance in S. cerevisiae have been demonstrated. The two loci show a high frequency of recombination with each other. 2. In the majority of OR strains examined the mutation was found to map at the O Ilocus. 3. Mapping studies have indicated that the O Iand O IIloci are either unlinked or very weakly linked to each other. Both of these loci also appear to be essentially unlinked from the -R I-R II-R IIIsegment of the mitochondrial genome specifying mitoribosomal functions. 4. Analysis of crosses involving mutants at either the O Ior O IIloci and a series of – petites variously deleted in known mitochondrial genes has demonstrated that the two oligomycin resistance loci O Iand O IIare separable. Assuming that the non deleted segment of the mitochondrial DNA is continuous, the results suggest the gene order -R I-R II-R III-O I-O II. 5. The rules applicable to the system and previously delineated from studies of the R I, R IIand R IIIloci in oligomycin sensitive strains have been shown to apply equally to strains carrying OR alleles at the O Iand O IIloci. Oligomycin resistance alleles at both the O loci, O Iand O II, are present in both + and – strains. 6. The presence of a nuclear gene(s) in a strain D6, influencing certain facets of the recombination process in both homosexual and heterosexual crosses has been demonstrated and this behaviour is superimposed upon, and clearly separable from the effects due to the mitochondrial gene .
Article
1. Mitochondria from aerobically grown wild-type yeast Saccharomyces cerevisiae, incubated anaerobically in a K+-containing medium are impermeable to protons. Permeability to protons was induced by uncoupler in the presence of valinomycin. Swelling in potassium acetate and energy-dependent K+ transport induced by valinomycin were similar to those of mammalian mitochondria except that the extent of the volume changes or of K+ uptake was considerably smaller with yeas mitochondria.2. Mitochondria isolated from a cytoplasmic respiration-deficient “petite” mutant were also quite impermeable to H+. As in wild-type yeast mitochondria, valinomycin enhanced the passive permeability for K+ in the mutant mitochondria and, together with an uncoupler, induced proton permeability. However, no ATP-dependent K+ uptake could be demonstrated.3. In contrast to wild-type yeast mitochondria, mitochondria from the respiration-deficient mutant exhibited neither uncoupler-sensitive 32Pi-ATP exchange nor a decrease in 8-anilino-1-naphthalene sulphonic acid, sodium salt (ANS) fluorescence on addition of ATP. It is concluded that although the permeability characteristics of the mutant mitochondria are similar to those of normal yeast mitochondria, the terminal segment of the energy-transfer system is rendered non-functional by the cytoplasmic mutation.
Article
1. An ATPase which is activated by phospholipids and inhibited by oligomycin, has been purified from beef heart submitochondrial particles using affinity chromatography. Phospholipid and detergent are removed by washing the enzyme with a solution of serum albumin while it is attached to the biospecific adsorbent.2. The ATPase is activated up to 18-fold by lysolecithin and to a smaller extent by cardiolipin, phosphatidylinositol and phosphatidylethanolamine. The amount required of each of these phospholipids to give half-maximal activation is apparently inversely related to the number of fatty acid chains in the lipid. Lecithin, which is a poor activator of the ATPase, competitively inhibits the activation by cardiolipin.3. The activation of the ATPase consists of an increase in both the maximal velocity of the reaction and the affinity for substrate ATP. The pH optimum of the reaction is not influenced by the charge of the lipid.4. Arrhenius plots of ATPase activated with lysolecithin show a transition to a higher activation energy at temperatures below 19 °C. The sensitivity of the lysolecithin-activated enzyme to oligomycin is markedly reduced below the same temperature. With cardiolipin the transition is observed at 13 °C.5. ADP, Mg2+ and to a smaller extent ATP, Mg2+ enhance the activation of ATPase by suboptimal amounts of phospholipid.
Article
1. The naturally occurring mitochondrial ATPase inhibitor inhibits the mitochondrial ATPase (F1) non-competitively.2. The interaction between inhibitor and inhibitor-depleted F1 or submitochondrial particles is diminished when the ratio of ATP/ADP is low or when energy is generated by substrate oxidation.3. The dissociation of the inhibitor from coupled Mg-ATP particles is promoted when substrates are being oxidized. This results in the appearance of a large uncoupler-stimulated ATPase activity. Activation of the uncoupler-stimulated ATPase activity is also achieved by incubation of the particles with ADP.4. The ATPase activity of Mg-ATP particles is determined by the turnover capacity of F1. When endogenous inhibitor is removed, energy dissipation becomes the rate-limiting step. This energy dissipation can be activated by an uncoupler.5. Evidence is presented for the existence of a non-inhibited intermediate F1-inhibitor complex.
1.1. A separation of polypeptides is reported based on polyacrylamide-gel electrophoresis in the presence of the cationic detergent cetyltrimethylammonium bromide. Like sodium dodecylsulphate electrophoresis, it shares the advantages of dissociability coupled with a linear relationship between the migration velocity and the logarithm of the molecular weight at least when applied to the simpler hydrophilic proteins. Polypeptide chain molecular weights can thus be estimated with considerable accuracy.2.2. More complex membrane proteins generally produced fewer bands with the cationic detergent than with sodium dodecylsulphate. Studies with the oligomycinsensitive ATPase protein indicate that this is due principally to an exclusion from the gel of the subunits with greatest hydrophobicity.
Article
A simple procedure for the purification of Mg2+-stimulated ATPase of Escherichia coli by fractionation with poly(ethylene glycols) and gel filtration is described. The enzyme restores ATPase-linked reactions to membrane preparations lacking these activities. Five different polypeptides (alpha, beta, gamma, delta, epsilon) are observed in sodium dodecyl sulfate electrophoresis. Freezing in salt solutions splits the enzyme complex into subunits which do not possess any catalytic activity. The presence of different subunits is confirmed by electrophoretic and immunological methods. The active enzyme complex can be reconstituted by decreasing the ionic strength in the dissociated sample. Temperature, pH, protein concentration, and the presence of substrate are each important determinants of the rate and extent of reconstitution. The dissociated enzyme has been separated by ion-exchange chromatography into two major fragments. Fragment IA has a molecular weight of about 100000 and contains the alpha, gamma, and epsilon polypeptides. The minor fragment, IB, has about the same molecular weight but contains, besides alpha, gamma, and epsilon, the delta polypeptide. Fragment II, with a molecular weight of about 52000, appears to be identical with the beta polypeptide. ATPase activity can be reconstituted from fragments IA and II, whereas the capacity of the ATPase to drive energy-dependent processes in depleted membrane vesicles is only restored after incubation of these two fractions with fraction IB, which contains the delta subunit.
Article
The soluble beef heart mitochondrial ATPase (F1) contains eight sulfhydryl groups and two disulfide bonds. N-Ethylmaleimide has been used to radioactively label the sulfhydryl groups before and after cleavage of the disulfide bonds by dithiothreitol. After subjecting the labeled protein to polyacrylamide gel electrophoresis in sodium dodecyl sulfate and measuring radioactivity in each of the separated subunits the location of all the sulfhydryl groups and the disulfide bonds may be specified. The conclusions are supported by direct examination of depolymerized, unreduced, enzyme by polyacrylamide gel electrophoresis. The results also indicate that current ideas regarding the overall subunit structure of this enzyme may be incorrect, and this is discussed in light of new data presented here.
Article
1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity. The inhibitor activity depends on the growth conditions of the yeast cells. It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane. 2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity. The minimal molecular weight calculated from amino acid composition is close to 7500. Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000. 3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties. It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6). In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles. 4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium. 5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine. It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M. The number of binding sites is of the order of 0.1 nmol/mg protein.
Article
Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecyl-sulfate-gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F1 ATPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex.
Article
Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class l and class 2. Class l mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, ‘1799’, tetrachlorotrifluoromethyl benzimidazole, carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to triethyl tin and the uncoupling agent ‘1799’ [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 μM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex.
Article
A polypeptide with a molecular weight of 8 500 (HP 8 500) was isolated from the mitochondrial membrane of the nuclear mutant cni-1 of Neurospora crassa. This mutant is characterized by a cyanide-insensitive respiration and by a deficiency in the cytochromes aa3 and b. The polypeptide is synthesized on mitochondrial ribosomes. It has an extremely hydrophobic character; it is insoluble in aqueous media in the absence of sodium dodecylsulfate and is soluble in acid chloroform/methanol. It lacks histidine. The polar amino acids lysine, arginine, aspartic acid, glutamic acid, serine and threonine make up only 25% of the total amino acids on a mole-percent basis. The N-terminal amino acid is tyrosine. The possible function of this polypeptide in the mitochondrial membrane is discussed.